Enhancement of in vitro cytopathogenicity by Acanthamoeba spp. following acquisition of bacterial endosymbionts

Approximately one in five isolates of Acanthamoeba spp. recovered from clinical and environmental sources are found to harbor obligate, uncultured bacterial endosymbionts of unknown clinical significance. To investigate their possible role in amoebic pathogenesis, four uninfected amoebic strains were exposed to four different endosymbionts, from which 12 stably-infected host-symbiont pairs resulted. Standardized inocula of amoebae with and without endosymbionts were placed on fibroblast monolayers to examine for cytopathic effects (CPEs). Eight to 10 days were required for monolayer effacement by endosymbiont-free amoebae; 5–8 days for amoebae containing Gram-negative rod endosymbionts; and 3 days for two amoebic isolates infected with a Chlamydia -like endosymbiont. All endosymbiont-infected amoebae produced a statistically significant enhancement in CPEs in comparison to uninfected amoebae; endosymbionts alone on monolayers produced no CPEs. This report provides evidence that obligate bacterial endosymbionts are able to enhance amoebic pathogenic potential in vitro by some as-yet unknown mechanism.     

Changes in sensitivity to anoxia of the cardiac action potential plateau during chick embryonic development.

The effects of anoxia on cardiac action potentials were studied at different stages of development of embryonic chick heart. The plateau phase of the action potential was markedly depressed by anoxia in old (15–16 days old) embryonic hearts without any significant change in other configurations of the action potential. Raising the concentration of glucose in the external fluid prevented the shortening of the action potential plateau by anoxia, and, conversely, a further reduction was observed in glucose-free media. In young (3–4 days old) embryonic hearts, the shortening of the action potential plateau was not produced by anoxia, but was produced by a combination of anoxia and glucose deprivation. When the action potential was shortened by anoxia in old hearts and by anoxia plus glucose deprivation in young hearts, isoproterenol (10^?5M), dibutyryl cyclic 3′,5′-adenosine monophosphate (dBcAMP: 1 mM) plus aminophylline (1 mM), and calcium ion (3–6 mM), partially reversed the shortened action potential in old hearts, but did not produce any prolongation in the young hearts. Therefore, the cation channels responsible for the action potential plateau in young hearts may be pharmacologically different from those in old hearts. The differences in action potential plateau between young and old hearts were discussed in relation to dependence upon energy.     

Symbiosis in the green leafhopper,Cicadella viridis (Hemiptera,Cicadellidae). Association in statu nascendi?

The green leafhopper, Cicadella viridis lives in symbiotic association with microorganisms. The ultrastructural and molecular analyses have shown that in the body of the C. viridis two types of bacteriocyte endosymbionts are present. An amplification and sequencing of 16S rRNA genes revealed that large, pleomorphic bacteria display a high similarity (94–100%) to the endosymbiont ‘Candidatus Sulcia muelleri’ (phylum Bacteroidetes), whereas long, rod-shaped microorganisms are closely related to the γ-proteobacterial symbiont Sodalis (97–99% similarity). Both endosymbionts may be harbored in their own bacteriocytes as well as may co-reside in the same bacteriocytes. The ultrastructural observations have revealed that the Sodalis-like bacteria harboring the same bacteriocytes as bacterium Sulcia may invade the cells of the latter. Bacteria Sulcia and Sodalis-like endosymbionts are transovarially transmitted from one generation to the next. However, Sodalis-like endosymbionts do not invade the ovaries individually, but only inside Sulcia cells. Apart from bacteriocyte endosymbionts, in the body of C. viridis small, rod-shaped bacteria have been detected, and have been identified as being closely related to γ-proteobacterial microorganism Pectobacterium (98–99% similarity). The latter are present in the sheath cells of the bacteriomes containing bacterium Sulcia as well as in fat body cells.     

Protein synthesis by intracellular symbionts in two closely interrelated aphid species

An aphid endosymbiont in vivo synthesizes symbionin almost exclusively which is not produced in vitro by the same symbiont. While symbionin produced by the endosymbiont of the pea aphid is an acidic protein with a molecular weight of 63,000, that by the symbiont of the kondo aphid, the closest relative to the former, is a distinct, less acidic, molecule. While the two endosymbionts in vivo in old insects synthesize about 11 protein species in common, they produce many different proteins when incubated extracellularly.     

Schistosomatium douthitti: effects of Lymnaea catascopium age on susceptibility to infection

Laboratory-reared Lymnaea catascopium snails (1–269 days old) were exposed individually to different numbers of Schistosomatium douthitti miracidia. Increasing the exposure dosage from 3 to 10 miracidia generally increased infection rates, in some age classes up to 100%. Successful re-exposure of snails not infected after a primary exposure was possible. Neonatal snails were least likely to become infected, primarily because miracidia were not attracted to them. Snails 12–55 days old were most susceptible to infection. Miracidia were readily attracted to these snails, and many were ingested and subsequently penetrated the host esophageal wall. Miracidial penetration of external snail surfaces was rare. Susceptibility of older snails (65–269 days) progressively declined with age. Many miracidia were entangled and immobilized in mucus produced by these snails, and fewer were ingested. No conspicuous host cellular responses to mother sporocysts were observed in any of the snails sectioned. A comparison of susceptibility of deliberately stunted snails and comparably aged controls of normal size indicated that the former were more susceptible.     

Strain-Specific Proteins of Symbiont-Containing Amoeba proteus Detected by Two-Dimensional Gel Electrophoresis1

Two-dimensional gel electrophoresis was used to compare the protein composition of normal (D strain) and symbiont-containing (xD strain) A. proteus. Over 500 different peptide spots could be identified on a typical gel of cytosol proteins. In addition to differences in a few minor peptides, one prominent polypeptide with an isoelectric point of about 5.5 and a molecular weight of about 29,000 was present in the cytoplasm of symbiont-containing amoebae and in the endosymbionts but not in D-strain amoebae. When endosymbionts were removed from xD-strain amoebae, the xD-specific polypeptide also disappeared, suggesting that it is produced by the endosymbionts.     

The effect of age on enolase turnover in the free-living nematode, Turbatrix aceti.

The rates of synthesis and degradation of enolase and total soluble proteins slow with age in the free-living nematode, Turbatrix aceti. The half-lives are 73 and 58 h for soluble protein and enolase, respectively, in young organisms (5 days old). The respective figures are 163 and 161 h for old organisms (22–30 days old). Similar slowing of protein turnover occurs when the organisms are aged by a repeated screening procedure which avoids the use of fluorodeoxyuridine, an inhibitor of DNA synthesis normally added to aging cultures to obtain synchrony. The results support the idea that slowed protein turnover may be responsible for the formation of altered enzymes in old organisms.     

Degradation of chlorpyriphos and polyethylene by endosymbiotic bacteria from citrus mealybug

Chlorpyriphos is one of the major organophosphorus pesticides used widely to control a range of insect pests across several crops. This insecticide is hazardous to the environment and toxic to mammals, thus, it is essential to remove the same from the environment. Similarly, use of polythene is also increasing day by day. Therefore, it is highly important to identify ways to degrade chlorpyriphos and other pesticides from the environment. We studied the degradation of chlorpyriphos and polyethylene by Citrus mealybug (Planococcus citri) bacterial endosymbionts such as Bacillus licheniformis, Pseudomonas cereus, Pseudomonas putida and Bacillus subtilis. This investigation revealed that bacterial endosymbionts use the polythene as a source of carbon and solubilize them by their enzymatic machinery. The degradation of polyethylene by endosymbionts showed a significant reduction in weight of polyethylene sheet after 15, 30 and 45 days of treatment. The SEM images showed localized degradation of the polyethylene around the bacterial cells in the biofilm. Further, the tensile strength (percentage elongation) was significantly reduced after 45 days of incubation. The weight of paraffin wax showed significant reduction in B. cereus. A significant reduction in total amount of chlorpyriphos in soil was observed at an interval of 7, 14 and 21 days after treatment by the bacterial isolates. Among the bacteria, B. cereus and P. putida were found to be most effective. The results from this study show that endosymbionts can be significantly implicated in degrading chlorpyriphos and polyethylene from the environment.     

Molecular identification of rickettsial endosymbionts in the non-phagotrophic volvocalean green algae

Background

The order Rickettsiales comprises Gram-negative obligate intracellular bacteria (also called rickettsias) that are mainly associated with arthropod hosts. This group is medically important because it contains human-pathogenic species that cause dangerous diseases. Until now, there has been no report of non-phagotrophic photosynthetic eukaryotes, such as green plants, harboring rickettsias.

Methodology/Principal Findings

We examined the bacterial endosymbionts of two freshwater volvocalean green algae: unicellular Carteria cerasiformis and colonial Pleodorina japonica. Epifluorescence microscopy using 4′-6-deamidino-2-phenylindole staining revealed the presence of endosymbionts in all C. cerasiformis NIES-425 cells, and demonstrated a positive correlation between host cell size and the number of endosymbionts. Strains both containing and lacking endosymbionts of C. cerasiformis (NIES-425 and NIES-424) showed a >10-fold increase in cell number and typical sigmoid growth curves over 192 h. A phylogenetic analysis of 16 S ribosomal (r)RNA gene sequences from the endosymbionts of C. cerasiformis and P. japonica demonstrated that they formed a robust clade (hydra group) with endosymbionts of various non-arthropod hosts within the family Rickettsiaceae. There were significantly fewer differences in the 16 S rRNA sequences of the rickettsiacean endosymbionts between C. cerasiformis and P. japonica than in the chloroplast 16 S rRNA or 18 S rRNA of the host volvocalean cells. Fluorescence in situ hybridization demonstrated the existence of the rickettsiacean endosymbionts in the cytoplasm of two volvocalean species.

Conclusions/Significance

The rickettsiacean endosymbionts are likely not harmful to their volvocalean hosts and may have been recently transmitted from other non-arthropod organisms. Because rickettsias are the closest relatives of mitochondria, incipient stages of mitochondrial endosymbiosis may be deduced using both strains with and without C. cerasiformis endosymbionts.     

Ultrastructure of the rickettsial endosymbiont “MIDORIKO” in the green alga Carteria cerasiformis as revealed by high-pressure freezing and freeze-substitution fixation

Bacterial endosymbionts belonging to the family Rickettsiaceae were recently identified in the unicellular green alga Carteria cerasiformis, providing the first molecular evidence of rickettsial endosymbionts within photosynthetic eukaryotes. However, previous morphological studies using transmission electron microscopy (TEM) with conventional chemical fixation did not demonstrate whether the endosymbionts of C. cerasiformis have the diagnostic characteristics of the family Rickettsiaceae. In this study, we observed the rickettsial endosymbionts “MIDORIKO” within C. cerasiformis cells by TEM with high-pressure freezing and freeze-substitution fixation. The rickettsial endosymbionts resided directly in the C. cerasiformis cytoplasm without engulfing or encompassing membranes or vacuoles. The endosymbionts had a Gram-negative cell envelope composed of outer and inner bilayer membranes. The thicknesses of the outer and inner leaflets of the bacterial cell wall were almost identical. These morphological characteristics are consistent with those of the genus Rickettsia, but the cell wall structure differed from that of the genus Orientia within the family Rickettsiaceae.     

Alteration with age of symbiosis of gene expression in aphid endosymbionts

Aphid endosymbionts in vivo in young hosts synthesized almost exclusively only one protein, symbionin. The synthesis of symbionin declined with age of the host and instead the endosymbiont began to express some of its own genes which were expressed in vitro but were repressed in vivo in young host. A prolonged treatment of young host with cycloheximide brought about a physiological state similar to that in old insect. Though in the very old insect symbionin was no longer produced by its endosymbiont, the host seemed to depend almost entirely upon the gene products of the endosymbiont.     

Population Dynamics and Growth Rates of Endosymbionts During Diaphorina citri (Hemiptera,Liviidae) Ontogeny

The infection density of symbionts is among the major parameters to understand their biological effects in host–endosymbionts interactions. Diaphorina citri harbors two bacteriome-associated bacterial endosymbionts (Candidatus Carsonella ruddii and Candidatus Profftella armatura), besides the intracellular reproductive parasite Wolbachia. In this study, the density dynamics of the three endosymbionts associated with the psyllid D. citri was investigated by real-time quantitative PCR (qPCR) at different developmental stages. Bacterial density was estimated by assessing the copy number of the 16S rRNA gene for Carsonella and Profftella, and of the ftsZ gene for Wolbachia. Analysis revealed a continuous growth of the symbionts during host development. Symbiont growth and rate curves were estimated by the Gompertz equation, which indicated a negative correlation between the degree of symbiont–host specialization and the time to achieve the maximum growth rate (t*). Carsonella densities were significantly lower than those of Profftella at all host developmental stages analyzed, even though they both displayed a similar trend. The growth rates of Wolbachia were similar to those of Carsonella, but Wolbachia was not as abundant. Adult males displayed higher symbiont densities than females. However, females showed a much more pronounced increase in symbiont density as they aged if compared to males, regardless of the incorporation of symbionts into female oocytes and egg laying. The increased density of endosymbionts in aged adults differs from the usual decrease observed during host aging in other insect–symbiont systems.     

Detection of an In Vivo 8 kD C-terminal Breakdown Product of D1-protein under Visible and UV-B Light

An 8 kD in vivo turnover product of D1 polypeptide was identified using a C-terminal specific anti-D1-antibody in thylakoids isolated from 5–10 days old seedlings of wheat (Triticum aestivum cv HD2329). Eight days old wheat seedlings grown under visible light were irradiated with UV-B (1 MW cm^?2) alone (0–5h) and Tricine SDS-PAGE was run of the isolated thylakoids. UV-B exposure of intact wheat leaves generates a fragment of 24 kD and concomitant increase in 8 kD fragment was also observed. From these results it is concluded that in wheat visible light induces the breakdown of D1 polypeptide into an 8 kD C-terminal thylakold bound fragment and UV-B stress results in an increase in 8 kD breakdown fragment, thus suggesting that visible light/UV-B has a common hot spot on D1 protein in wheat.     

Endosymbionts of Acanthamoeba Isolated from Domestic Tap Water in Korea

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the α-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.     

The biochemical composition of the larvae of two strains of Artemia salina (L.) reared on two different algal foods

A sequential carbohydrate, protein, and lipid method of analysis has been used to determine the biochemical composition of freshly hatched and 48-h old larvae of two strains of the brine shrimp Artemia salina (L.). During a 48-h starvation period the percentage of carbohydrates and lipids of freshly hatched larvae decreases whereas the ash content increases by 40–100%. When fed with dried Scenedesmus or dried Spirulina for 2 days after hatching the protein level of the larvae increases significantly and the relative increase in ash content is lower than in the case of starvation.Amino acid analyses of the algal food and the unfed and fed larvae did not show any change except for the absence (below detection) of methionine in the starved nauplii.The fatty acid pattern of 48-h old Artemia larvae is different from that of freshly hatched nauplii both in unfed and fed larvae; in the latter case it seems to be determined to a large extent by the fatty acid composition of the food.     

Domestication in the silver fox (Vulpes fulvus Desm): Changes in physiological boundaries of the sensitive period of primary socialization

The physiological boundaries of the sensitive period of primary socialization were studied in the silver fox (Vulpes fulvus Desm). A total of 273 farm-bred foxes from 59 litters were observed from 1976 to 1978; pups were produced by vixens from two populations, one selected for domesticated behaviour and the other unselected. Results indicate that the age when the eyes are fully open, when the response to sound first appears and when exploratory behaviour is first shown in strange surroundings is 3 weeks, on average. The age when the socialization period starts appears to be 20–25 days old. The optimum period of the formation of primary social bonds appears to be 30–35 days, when maximum exploration in a novel situation is shown. The 40–45 days period appears to be the upper boundary of primary socialization in unselected foxes because pups show fear in response to novel stimuli, which prevents exploration. In pups from the population of domesticated foxes, the sensitive period of socialization is prolonged to over 60–65 days old.     

Primary infection with mouse-derived cysticercoids of Hymenolepis nana prepared from baby or adult mice and secondary infections with eggs or cysticercoids

Oral infection with mouse-derived cysticercoids (cysts) of Hymenolepis nana on day 0 did not make the 5 ± 1 week old mouse host immune to egg challenge by day 7 of the prepatent period, although the number of cyst-derived tapeworms was 1000 times greater than that of egg-derived tapeworms sufficient to make the host immune by day 7. Neither cysts recovered from immunologically competent 5 ± 1 week old donor mice, which should have become immune within 2 days of egg inoculation, nor those from incompetent 5–7 day-old baby mice given eggs when 0–2 days old made the host immune. The time course of differentiation of cysts in baby mice was not different from that in 5 ± 1 week-old mice. Mice infected twice with cysts on days 0 and 4 did not become immune either. Rapid protective immunity against egg challenge was acquired by inoculation exclusively with eggs but not with cysts. Apparently cysts differ from oncospheres in their immunogenicity. The importance of cysts for analysing the mouse—H. nana system from the immunological point of view is discussed.     

Diversity and geographic variation of endosymbiotic bacteria in natural populations of the pea aphid (Acyrthosiphon pisum) in China

Bacterial symbionts in aphids are known to benefit the insect host and associated with aphid’s ecological adaptation. The pea aphid (Acyrthosiphon pisum), an important legume pest worldwide, carries at least eight endosymbionts, providing a model system to study insect–bacteria interactions. However, species diversity and geographic variations of endosymbionts are unknown in Chinese populations; therefore, we characterized symbiont communities and diversity of 27 pea aphid samples from 13 geographic populations of China. Via amplicon high-throughput sequencing and diagnostic PCR, we found that bacterial communities of Chinese populations were dominated by Proteobacteria and Firmicutes. Among eight known endosymbionts, five (Buchnera, Serratia, Hamiltonella, Regiella, and Rickettsia) were detected by both methods, with a specific geographical distribution. The obligate symbiont, Buchnera, was present in all aphid samples, while the four facultative symbionts showed a significant geographic variation. Each population was randomly infected with distinct endosymbionts, ranging from three to five species. Serratia and Rickettsia showed relatively higher abundance in central regions of China, Regiella was predominant in eastern and western China, whereas Hamiltonella showed an extremely low abundance and was absent in four populations. Samples grouped by altitudes showed a significant diversity difference, whereas there was no significant difference between red and green body colors. Bacterial community structures of the Chinese pea aphid populations were mainly influenced by environmental factors, other than body colors. These data can guide the development of potential biocontrol techniques against this aphid.     

Experimental observations on the transmission of Schistosoma margrebowiei miracidia

Evans N. A. 1985. Experimental observations on the transmission of Schistosoma margrebowiei miracidia. International Journal for Parasitology15: 361–364. The survival and infectivity characteristics of Schistosoma margrebowiei miracidia at a temperature of 26°C are described. The maximum survival time and the time to 50% survival were approx. 13 and 9.5 h respectively. Miracidial infectivity toward Bulinus natalensis was relatively constant for the first 4 h of life but it then declined steadily to zero after 11–12 h. Snails exhibited age-dependent differences in their susceptibility to infection, individuals being most susceptible when 4 days old. Increases in snail age beyond 1 week were generally accompanied by an increased level of resistance to infection. Exposure of neonate (< 2 days old) snails produced high levels of mortality and a very low proportion of the survivors were infected at the time of parasitological examination.